- Email: [email protected]
ALOX5 promoter genotype and response to montelukast in moderate persistent asthma
ARTICLE IN PRESS Respiratory Medicine (2008) 102, 857–861
ALOX5 promoter genotype and response to montelukast in moderate persistent asthma Juan J. Telleriaa,b,, Alfredo Blanco-Quirosa,b, David Varillasa, Alicia Armentiac, Isabel Fernandez-Carvajala,b, M. Jesus Alonsoa,b, Ignacio Diezd a
Institute of Biology and Molecular Genetics (IBGM/CSIC), University of Valladolid, Valladolid, Spain Department of Pediatrics, University of Valladolid, Valladolid, Spain c University Hospital Rio Hortega, Valladolid, Spain d Hospital Txagorritxu, Vitoria, Spain b
Received 6 December 2007; accepted 13 January 2008 Available online 12 March 2008
KEYWORDS Asthma; Pharmacogenetics; Leukotrienes; Response rate; 5-Lipoxygenase
Summary Background: It was hypothesized that asthmatic patients with mutant alleles in the leukotriene pathway should not respond to leukotriene receptor antagonists and the concept of a tailored treatment is increasingly supported. Methods: Sixty-one patients (mean age 24.9 years, range 14–52) with moderate persistent asthma were clinical and immunological assess prior and after a 6-month treatment with montelukast. Tandem repeat polymorphisms were genotyped in the promoter (147 to 176) of 5-lipoxygenase gene (ALOX5). Results: Thirty-two patients (52.5%) were homozygous for the five repeats allele; 17 (27.9%) were heterozygous (4/5 repeats) and 12 (19.7%) were homozygous for 4/4 repeats. After the montelukast treatment decrease number of asthma exacerbations, improvement of FEV1 and decreased use of b2 agonists was observed in patients with 5/5 or 4/5 repeats. Conversely, the patients with 4/4 repeats genotype did not modify these data after treatment. Conclusions: It was confirmed that ALOX5 promoter polymorphisms have a clear influence in montelukast response in atopic moderate persistent asthma patients. The genetic study could identify those patients most likely to respond to montelukast. & 2008 Elsevier Ltd. All rights reserved.
Abbreviations: 5-LO, 5-lipoxygenase; ALOX5, 5-lipoxygenase gene; CysLT, cysteinyl leukotriene; FEV1, forced expiratory volume in the 1 s; LT, leukotriene; LTRAs, leukotriene receptor antagonists. Corresponding author at: Instituto de Biologı´a y gene ´tica Molecular (IBGM), C/ Sanz y Fore ´s s.n., 47003 Valladolid, Spain. Tel.: +34 636 592 478; fax: +34 983 183 812. E-mail address: [email protected] (J.J. Telleria). 0954-6111/$ - see front matter & 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.rmed.2008.01.011
ARTICLE IN PRESS 858
Introduction Leukotrienes (LTs) are a family of lipid mediators with a crucial role in the inflammation.1 5-lipoxygenase (5-LO) catalyses the conversion of arachidonic acid into LTA4, that afterwards will be transformed to LTB4 or cysteinyl-LTs (CysLTs). In this process, 5-LO and its activating protein (FLAP) act as limiting enzymes.2 There are two types of LTs: LTB4, a potent chemotactic mediator of inflammation, and the CysLTs group, including LTC4, LTD4 and LTE4, all of them powerful bronchoconstrictors, therefore playing a key role in the pathophysiology of asthma and other inflammatory diseases. The development of LT receptor antagonists (LTRAs), such as montelukast or zafirlukast, represented an important step towards the control of those diseases. Due to their anti-inflammatory and anti-bronchoconstrictor properties, these drugs improve lung function parameters, such as forced expiratory volume in the 1 s (FEV1) or peak flow, reduce bronchial hyperresponsiveness and decrease the number of asthma exacerbations, tapering the use of inhaled corticosteroids which are the cornerstone of asthma control. However, the patients show a widely variable response to LTRAs treatment, fact that has been attributed to genetic factors in 60% of the cases.3 The variance in genes coding enzymes of the LTs synthesis cascade, such as 5-LO, FLAP and LTC4 synthase, could be the explanation of this different response to LTRAs.4 5-LO is coded by the ALOX5 gene that has a tandem repeat polymorphism within the transcription factor-binding region at position 147 to 176, of its promoter, a region which is able to regulate gene transcription. Human cell lines containing other than the five tandem Sp1 repeats allele in the promoter region of ALOX5 seems to show a low activity in the promoter-reporter activity.5 A previous study performed with ABT-761, a direct inhibitor of 5-LO, showed that patients with at least one wild-type allele (five tandem repeats) had better response to this drug than patients bearing only mutant alleles (other than five repeats).6 Based on this finding, it was hypothesized that LTs are not responsible for airway obstruction in the patients who fail to respond to ALOX5 inhibition. According to this model, asthmatic patients with only mutant alleles should not respond to LTRAs drugs such as montelukast.6 The present paper shows the influence of the polymorphism repeats variance of ALOX5 gene on the response to the treatment with montelukast, being evaluated by FEV1, number of asthma attacks and need of rescue medication.
J.J. Telleria et al. demand. Despite this treatment, they had suffered episodes of cough and/or wheezing during the last 2 years. No patient was treated with oral steroids. Treatment with montelukast was initiated as an add-on therapy given the inefficient control of the disease. The following data were collected prior and after the 6-month treatment period with montelukast:
FEV1 in % of predicted. Number of puffs of short acting b2-agonist per day (puffs/ 24 h) during the last month.
Number of exacerbations. The exacerbations were defined as a decrease in peak expiratory flow rate in the morning of at least 25% compared with baseline.8 Blood sample was taken at the time of treatment instauration and sent to the laboratory for genotyping. After 6-month treatment period with montelukast, current FEV1, consumption of b2 agonists and exacerbations in the last month were recorded. All clinical data were recorded by the same physician ignoring the genotype of the patients. Controls: Sixty-one matched control patients were genotyped in order to compare their allelic and genotypic frequencies with the group of patients. Genotyping: The ALOX-5 repeat polymorphisms study was performed by hot-start polymerase chain reaction of an amplicon containing the polymorphic region. The reaction generates a 274 bp fragment (wild-type allele). Given that GC rich region polymorphisms are difficult to genotype, the GC-Rich PCR System (Roche Diagnostic GmbHs, Manheim, Germany) was used. The amplicons were analysed in 6% polyacrylamide gels (Figure 1). The primers used were: forward 50 AGG AAC AGA CAC CTC GCT GAG GAG AG 30 ; and reverse 50 GAG CAG CGA GCC CCG GGA GCC TCG GC 30 . The PCR protocol was: initial denaturalization 5 min at 94 1C followed by 30 cycles of 30 s at each of the following temperatures: 94, 64 and 72 1C. A final incubation of 7 min was carried out at 72 1C.9 In order to avoid misinterpretations, the genotype were analysed blind with regard to clinical data. Data analysis: Improvements in FEV1, asthma exacerbations (last month) and use of b2 agonist (puffs/day, last month) achieved during the treatment period were compared between patients homozygous for the allele with four tandem repeats within ALOX5 promoter (4/4) and patients with other genotypes (5/5 and 5/4). Two-sided Wilcoxon test was used to establish the significance of differences
Patients and methods The trial was designed as a prospective study and the aim was to study the response to montelukast in atopic asthmatic patients according to their ALOX-5 genotype. The Ethical Committee of the Faculty of Medicine of Valladolid approved the study. Patients: A total of 61 patients (29 males) with a mean age of 24.9 years (range 14–52) were included in the study. All patients suffered moderate persistent asthma according to the GINA guidelines,7 had serum IgE levels greater than 100 IU/mL, one or more positive prick test to aero-allergens and received budesonide (500 mg/12 h) and b2 agonist on
Figure 1 ALOX-5 genotyoing. Line 1: known 4/5 heterozygote used as a control. Line 2: molecular weight marker. Line 3: sample with genotype 5/4. Lines 4, 5, 7 and 8: samples with genotype 5/5. Lines 6 and 9. samples with genotype 4/4.
ARTICLE IN PRESS ALOX5 genotype and response to montelukast between the 4/4 tandem repeats and other groups (4/5 and 5/5). This test was chosen because it is more powerful than parametric tests for small groups.
Results Thirty-two patients (52.5%) were homozygous for the five repeats allele; 17 (27.9%) were heterozygous, and the remaining 12 (19.7%) were homozygous for the 4/4 repeats. In the control group, the distribution was as follows: 42 (68.8%) five repeats homozygous, 12 (19.7%) heterozygous and 7 (11.5%) 4/4 homozygous. The number of carriers of the allele with four repeats was higher in the group of asthma patients, but the difference did not reach statistical significance with p ¼ 0.1712. Before treatment, no clinical differences were found among the three groups (Table 1). Conversely, the values obtained after the 6-month treatment period with montelukast were statistically significants, the worst data for all variables were found in the group 4/4 tandem repeats homozygous group. The response of heterozygous (4/5 repeats) patients was similar to the response of reference group (5/5 repeats), except for the b2-agonists use, which show intermediate values between both homozygous groups, although clearly better than those of the 4/4 patients (Table 1). Given that patients with genotypes 5/5 and 4/5 showed similar response to montelukast, we grouped together these patients and compared their response with the group of patients homozygous 4/4. There were neither demographic (age and sex) differences nor regarding to serum IgE levels or sensitisation to aero-allergens (pollen and dust mites) measured by means of prick test among both genotypic groups (Table 2). Moreover, both compared groups showed similar clinical features (FEV1, number of puffs of acting b2-agonist per day during the last month and number of exacerbations) before therapy (Figure 2 and attached table). After the LTs treatment period, patients with 4/4 repeats allele had 1.88 (SD ¼ 0.92) rate of asthma exacerbations in Table 1
859 the last month, with a mean reduction of 1.33 (SD ¼ 1.22), whereas for cases with at least one five repeat allele was 0.4 (SD ¼ 0.21) exacerbations with a reduction of 4.41 (SD ¼ 2.76), a very significant difference (p ¼ 0.001). The change in FEV1 was also more favourable in the group with at least a five repeats: 93% (SD ¼ 7.0) with an increase of 8.6% (SD ¼ 7.61) versus 80% and reduction of 1.4% (SD ¼ 8.04) (p ¼ 0.0006). Finally, the 4/4 repeats group needed more b2 rescue medication: 5.1 (SD ¼ 2.84) with a reduction of 0.55 (SD ¼ 1.66) versus 2.9 (SD ¼ 2.77) and a reduction of 4.3 (SD ¼ 3.42) in the 5/5 repeats group (p ¼ 0.0011).
Discussion The efficacy of asthma treatment depends on the control of environmental factors and also on pharmacological therapy. Nowadays, increasing evidence is supporting the concept of a tailored treatment for each patient. To reach this objective, an exact knowledge of genetic influences on drug response would be necessary.10,11 It is estimated that up to 70% of the variability of the response to asthma treatment is due to genetic conditions. Some authors previously reported the influence of the A-444C SNP of the LTC4 synthase gene to the response to CysLTR inhibitors,12,13 and a more recent study had discovered a better response to montelukast in patients bearing the less frequent genotypes of the MRP1 (rs119774) and ALOX5 (rs2115819).14 The allele with five tandem repeats (wild type) was the most frequently found in our study in agreement to other reports,6,9 the allelic frequency was 0.664 and the frequency of homozygous patients was 0.525. This polymorphism had been associated to some asthma phenotypes such as the severity,15 but never to asthma susceptibility. In addition to five and four repeats, three and six repeat alleles have been occasionally reported in some papers, but we did not find them in the present study. Prior to the LTRAs treatment period no significant difference was present with respect to FEV1, b2 agonist use, and exacerbation rate, between patients carrying or not the five repeats allele. Conversely, after the 6-month
Severity of asthma before and after 6-months treatment with montelukast.
ALOX5 genotype
(5/5) (n ¼ 32)
(5/4) (n ¼ 17)
(4/4) (n ¼ 12)
Sex distribution (M/F) Age (SD) IgE in KUI/L (SD) IgE rank
15/17 27.0 (13.8) 649.8 (473.3) 180–2300
8/9 27.9 (13.19) 556.8 (554.3) 290–2000
6/6 23.3 (8.7) 622.2 (511.7) 240–2000
Exacerbations in the last month mean (SD) Before 4.7 (2.7) After 0.4 (0.2)
5.0 (2.5) 0.4 (0.2)
3.22 (1.0) 1.88 (0.9)
FEV1 mean (SD) Before After
82% (8.1) 93% (7.3)
85% (7.0) 92% (6.9)
81% (13.5) 80% (9.8)
Puffs/24 h mean (SD) Before After
6.9 (3.0) 2.2 (1.8)
7.8 (3.0) 4.0 (3.9)
6.1 (1.6) 5.1 (2.8)
ARTICLE IN PRESS 860
J.J. Telleria et al.
Table 2 Age and sex distribution, IgE levels and sensitisation to aerorallergens of patients homozygous 4/4 and patients with at least one allele with five tandem repeats within the transcription factor-binding region of ALOX5 gene. Male/female
Age
IgE (IU/mL)
Prick test Pollen
Dust mites
Both
4/4 Mean SD
6/6
23.33 8.75
622.22 511.68
10 (83.3%)
3 (25%)
1 (8.3%)
5/4+5/5 Mean SD
23/26
27.21 13.23
625.34 479.13
44 (89.8%)
11 (22.4%)
6 (12.2%)
Before therapy Exacerbations
7
100%
P=n.s.
6
95%
5
90%
4
10 9 8 7 6 5 4 3 2 1 0
P=n.s.
85%
3 2
80%
1
75%
0
70% 5/5+5/4
B2 adrenergic Puffs / 24h
FEV1 (%)
4/4
5/5+5/4
4/4
P=n.s.
5/5+5/4
4/4
After therapy
7
B2 adrenergic Puffs / 24h
FEV1 (%)
Exacerbations 100%
P=0.001
6
95%
5
90%
4
P=0.0006
85%
3 2
80%
1
75%
0
70% 5/5+5/4
5/5+5/4
4/4
4/4
BEFORE THERAPY Exacerb. FEV1 (%) B2 / 24h 4/4
5/4+5/5
10 9 8 7 6 5 4 3 2 1 0
P=0.0011
5/5+5/4
4/4
AFTER THERAPY Exacerb. FEV1 (%) B2 / 24h
mean
3.22
81%
6.11
1.89
80%
5.56
SD
0.97
13%
1.62
0.93
10%
2.40
mean
4.76
83%
7.13
0.39
93%
2.71
SD
2.59
8%
3.00
0.59
7%
2.95
Figure 2
treatment with LTRAs, patients with at least one set of five repeats showed better clinical evolution outcomes than patients with 4/4 repeat alleles. In fact, among this last
group of patients, the average response was small or absent. The differences between the two groups were very high despite the small sample size.
ARTICLE IN PRESS ALOX5 genotype and response to montelukast In agreement with other studies,1,16 we found no differences between wild type homozygous (5/5 repeats) and heterozygous (4/5 repeats) suggesting that the wildtype allele would be dominant, at least for the studied phenotypes. Classically, three different mechanisms have been said to be involved in the influence of polymorphisms to the therapy response: (a) variations affecting drug metabolism, (b) variations related to mechanisms leading to adverse effects, and (c) variations in the therapeutic target or in the pathways activated by it.17 Our results are similar to other ones reported by Drazen et al.6 but they observed the correlation between the response to an ALOX5 inhibitor and the tandem repeat polymorphism in the promoter of the same gene. This fact is consistent with early studies supporting that addition and deletion variants in the ALOX5 gene promoter diminished the activity of promoter–reporter in vitro.5 Nevertheless, a later study supported that some mutant variants of the ALOX5 gene could also up-regulate the expression of this gene.14 ALOX-5 and montelukast are not directly related, since 5-LO initiates the LT synthesis pathway and montelukast is an antagonist of the receptors on which final products of the pathway, namely the CysLTs, exert their actions. Bearing in mind that ALOX-5 repeat polymorphism is a transcription factor binding site which modulates transcription of the reporter gene5 and thus the conversion of arachidonic acid into LTA4, it may be expected that patients with 4/4 repeat have a limited ability to initiate the cascade of LT synthesis and it could be hypothesized that in these patients LTs would probably not contribute much to the physiopathology of the asthma and thus their inhibition would be of very limited efficacy. Asthmatic patients could then be classified as LT-dependent, who would respond to LT pathway inhibitors, and LT-independent, who would not respond to those drugs. The importance of repeat polymorphism in the ALOX5 promoter on the response to LTRAs is confirmed. More polymorphisms related to the synthesis of LT could also be involved in the treatment response to LT inhibitors, but likely, the relative frequencies of those polymorphisms widely vary among different populations. From our data and given the frequency of allele 4 in our population, we should expect a ratio of bad responders to LT receptor inhibitors ranging between 12.3% and 18.9% (CI ¼ 95%). This ratio is not age or sex dependent, but could be higher in patients with moderate or severe asthma.15
Conflict of interest statement The authors declare that none of them have any financial or another sort of conflict of interest in relation to this study that may influence their interpretation of the results.
Acknowledgments This work has been partially sponsored by grants of Instituto de Salud Carlos III (PI05/1746) and Fundacion Sociedad Espan ˜ola de Alergia e Inmunologı´a Clı´nica (2005).
861
References 1. Drazen JM, Israel E, O’Byrne PM. Treatment of asthma with drugs modifying the leukotriene pathway. N Engl J Med 1999;340:197–206. 2. Woods JW, Evans JF, Ethier D, Scott S, Vickers PJ, Hearn L, et al. 5-lipoxygenase and 5-lipoxygenase-activating protein are localized in the nuclear envelope of activated human leukocytes. J Exp Med 1993;178:1935–46. 3. Drazen JM, Silverman EK, Lee TH. Heterogeneity of therapeutic responses in asthma. Br Med Bull 2000;56:1054–70. 4. Hall IP, Sayers Y. Pharmacogenetics and asthma: false hope or new dawn? Eur Resp J 2007;29:1239–45. 5. In KH, Asano K, Beier D, Grobholz J, Finn PW, Silverman EK, et al. Naturally occurring mutations in the human 5-lipoxygenase gene promoter that modify transcription factor binding and reporter gene transcription. J Clin Invest 1997;99:1130–7. 6. Drazen JM, Yandava CN, Dube L, Szczerback N, Hippensteel R, Pillari A, et al. Pharmacogenetic association between ALOX5 promoter genotype and the response to anti-asthma treatment. Nat Genet 1999;22:168–70. 7. Masoli M, Fabian D, Holt S, Beasley R, Global Initiative for Asthma (GINA) Program. The global burden of asthma: executive summary of the GINA Dissemination Committee report. Allergy 2004;59:469–78. 8. Pauwels RA, Lofdahl CG, Postma DS, Tattersfield AE, O’Byrne P, Barnes PJ, et al. Effect of inhaled formoterol and budesonide on exacerbations of asthma. N Engl J Med 1997;337:1405–11. 9. Sayers I, Barton S, Rorke S, Sawyer J, Peng Q, Beghe B, et al. Promoter polymorphism in the 5-lipoxygenase (ALOX5) and 5-lipoxygenase-activating protein (ALOX5AP) genes and asthma susceptibility in a Caucasian population. Clin Exp Allergy 2003;33:1103–10. 10. Leff AR. Regulation of leukotrienes in the management of asthma: biology and clinical therapy. Annu Rev Med 2001; 52:1–14. 11. Wechsler ME. Managing asthma in the 21st century: role of pharmacogenetics. Pediatr Ann 2006;35:664–9. 12. Szczeklik A, Mastalerz L, Nizankowska E, Sanak M. Myocardial ischemia possibly mediated by cysteinyl leukotrienes. J Allergy Clin Immunol 2002;109:572–3. 13. Sampson AP, Siddiqui S, Buchanan D, Howarth PH, Holgate ST, Holloway JW, et al. Variant LTC(4) synthase allele modifies cysteinyl leukotriene synthesis in eosinophils and predicts clinical response to zafirlukast. Thorax 2000;55(Suppl 2): S28–31. 14. Lima JJ, Zhang S, Grant A, Shao L, Tantisira KG, Allayee H, et al. Influence of leukotriene pathway polymorphisms on response to montelukast in asthma. Am J Respir Crit Care Med 2006;173: 379–85. 15. Kalayci O, Birben E, Sackesen C, Keskin O, Tahan F, Wechsler ME, et al. ALOX5 promoter genotype, asthma severity and LTC production by eosinophils. Allergy 2006;61:97–103. 16. Fowler SJ, Hall IP, Wilson AM, Wheatley AP, Lipworth BJ. 5-Lipoxygenase polymorphism and in-vivo response to leukotriene receptor antagonists. Eur J Clin Pharmacol 2002;58: 187–90. 17. Sayers I, Hall IP. Pharmacogenetic approaches in the treatment of asthma. Curr Allergy Asthma Rep 2005;5:101–8.
ALOX5 promoter genotype and response to montelukast in moderate persistent asthma Juan J. Telleriaa,b,, Alfredo Blanco-Quirosa,b, David Varillasa, Alicia Armentiac, Isabel Fernandez-Carvajala,b, M. Jesus Alonsoa,b, Ignacio Diezd a
Institute of Biology and Molecular Genetics (IBGM/CSIC), University of Valladolid, Valladolid, Spain Department of Pediatrics, University of Valladolid, Valladolid, Spain c University Hospital Rio Hortega, Valladolid, Spain d Hospital Txagorritxu, Vitoria, Spain b
Received 6 December 2007; accepted 13 January 2008 Available online 12 March 2008
KEYWORDS Asthma; Pharmacogenetics; Leukotrienes; Response rate; 5-Lipoxygenase
Summary Background: It was hypothesized that asthmatic patients with mutant alleles in the leukotriene pathway should not respond to leukotriene receptor antagonists and the concept of a tailored treatment is increasingly supported. Methods: Sixty-one patients (mean age 24.9 years, range 14–52) with moderate persistent asthma were clinical and immunological assess prior and after a 6-month treatment with montelukast. Tandem repeat polymorphisms were genotyped in the promoter (147 to 176) of 5-lipoxygenase gene (ALOX5). Results: Thirty-two patients (52.5%) were homozygous for the five repeats allele; 17 (27.9%) were heterozygous (4/5 repeats) and 12 (19.7%) were homozygous for 4/4 repeats. After the montelukast treatment decrease number of asthma exacerbations, improvement of FEV1 and decreased use of b2 agonists was observed in patients with 5/5 or 4/5 repeats. Conversely, the patients with 4/4 repeats genotype did not modify these data after treatment. Conclusions: It was confirmed that ALOX5 promoter polymorphisms have a clear influence in montelukast response in atopic moderate persistent asthma patients. The genetic study could identify those patients most likely to respond to montelukast. & 2008 Elsevier Ltd. All rights reserved.
Abbreviations: 5-LO, 5-lipoxygenase; ALOX5, 5-lipoxygenase gene; CysLT, cysteinyl leukotriene; FEV1, forced expiratory volume in the 1 s; LT, leukotriene; LTRAs, leukotriene receptor antagonists. Corresponding author at: Instituto de Biologı´a y gene ´tica Molecular (IBGM), C/ Sanz y Fore ´s s.n., 47003 Valladolid, Spain. Tel.: +34 636 592 478; fax: +34 983 183 812. E-mail address: [email protected] (J.J. Telleria). 0954-6111/$ - see front matter & 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.rmed.2008.01.011
ARTICLE IN PRESS 858
Introduction Leukotrienes (LTs) are a family of lipid mediators with a crucial role in the inflammation.1 5-lipoxygenase (5-LO) catalyses the conversion of arachidonic acid into LTA4, that afterwards will be transformed to LTB4 or cysteinyl-LTs (CysLTs). In this process, 5-LO and its activating protein (FLAP) act as limiting enzymes.2 There are two types of LTs: LTB4, a potent chemotactic mediator of inflammation, and the CysLTs group, including LTC4, LTD4 and LTE4, all of them powerful bronchoconstrictors, therefore playing a key role in the pathophysiology of asthma and other inflammatory diseases. The development of LT receptor antagonists (LTRAs), such as montelukast or zafirlukast, represented an important step towards the control of those diseases. Due to their anti-inflammatory and anti-bronchoconstrictor properties, these drugs improve lung function parameters, such as forced expiratory volume in the 1 s (FEV1) or peak flow, reduce bronchial hyperresponsiveness and decrease the number of asthma exacerbations, tapering the use of inhaled corticosteroids which are the cornerstone of asthma control. However, the patients show a widely variable response to LTRAs treatment, fact that has been attributed to genetic factors in 60% of the cases.3 The variance in genes coding enzymes of the LTs synthesis cascade, such as 5-LO, FLAP and LTC4 synthase, could be the explanation of this different response to LTRAs.4 5-LO is coded by the ALOX5 gene that has a tandem repeat polymorphism within the transcription factor-binding region at position 147 to 176, of its promoter, a region which is able to regulate gene transcription. Human cell lines containing other than the five tandem Sp1 repeats allele in the promoter region of ALOX5 seems to show a low activity in the promoter-reporter activity.5 A previous study performed with ABT-761, a direct inhibitor of 5-LO, showed that patients with at least one wild-type allele (five tandem repeats) had better response to this drug than patients bearing only mutant alleles (other than five repeats).6 Based on this finding, it was hypothesized that LTs are not responsible for airway obstruction in the patients who fail to respond to ALOX5 inhibition. According to this model, asthmatic patients with only mutant alleles should not respond to LTRAs drugs such as montelukast.6 The present paper shows the influence of the polymorphism repeats variance of ALOX5 gene on the response to the treatment with montelukast, being evaluated by FEV1, number of asthma attacks and need of rescue medication.
J.J. Telleria et al. demand. Despite this treatment, they had suffered episodes of cough and/or wheezing during the last 2 years. No patient was treated with oral steroids. Treatment with montelukast was initiated as an add-on therapy given the inefficient control of the disease. The following data were collected prior and after the 6-month treatment period with montelukast:
FEV1 in % of predicted. Number of puffs of short acting b2-agonist per day (puffs/ 24 h) during the last month.
Number of exacerbations. The exacerbations were defined as a decrease in peak expiratory flow rate in the morning of at least 25% compared with baseline.8 Blood sample was taken at the time of treatment instauration and sent to the laboratory for genotyping. After 6-month treatment period with montelukast, current FEV1, consumption of b2 agonists and exacerbations in the last month were recorded. All clinical data were recorded by the same physician ignoring the genotype of the patients. Controls: Sixty-one matched control patients were genotyped in order to compare their allelic and genotypic frequencies with the group of patients. Genotyping: The ALOX-5 repeat polymorphisms study was performed by hot-start polymerase chain reaction of an amplicon containing the polymorphic region. The reaction generates a 274 bp fragment (wild-type allele). Given that GC rich region polymorphisms are difficult to genotype, the GC-Rich PCR System (Roche Diagnostic GmbHs, Manheim, Germany) was used. The amplicons were analysed in 6% polyacrylamide gels (Figure 1). The primers used were: forward 50 AGG AAC AGA CAC CTC GCT GAG GAG AG 30 ; and reverse 50 GAG CAG CGA GCC CCG GGA GCC TCG GC 30 . The PCR protocol was: initial denaturalization 5 min at 94 1C followed by 30 cycles of 30 s at each of the following temperatures: 94, 64 and 72 1C. A final incubation of 7 min was carried out at 72 1C.9 In order to avoid misinterpretations, the genotype were analysed blind with regard to clinical data. Data analysis: Improvements in FEV1, asthma exacerbations (last month) and use of b2 agonist (puffs/day, last month) achieved during the treatment period were compared between patients homozygous for the allele with four tandem repeats within ALOX5 promoter (4/4) and patients with other genotypes (5/5 and 5/4). Two-sided Wilcoxon test was used to establish the significance of differences
Patients and methods The trial was designed as a prospective study and the aim was to study the response to montelukast in atopic asthmatic patients according to their ALOX-5 genotype. The Ethical Committee of the Faculty of Medicine of Valladolid approved the study. Patients: A total of 61 patients (29 males) with a mean age of 24.9 years (range 14–52) were included in the study. All patients suffered moderate persistent asthma according to the GINA guidelines,7 had serum IgE levels greater than 100 IU/mL, one or more positive prick test to aero-allergens and received budesonide (500 mg/12 h) and b2 agonist on
Figure 1 ALOX-5 genotyoing. Line 1: known 4/5 heterozygote used as a control. Line 2: molecular weight marker. Line 3: sample with genotype 5/4. Lines 4, 5, 7 and 8: samples with genotype 5/5. Lines 6 and 9. samples with genotype 4/4.
ARTICLE IN PRESS ALOX5 genotype and response to montelukast between the 4/4 tandem repeats and other groups (4/5 and 5/5). This test was chosen because it is more powerful than parametric tests for small groups.
Results Thirty-two patients (52.5%) were homozygous for the five repeats allele; 17 (27.9%) were heterozygous, and the remaining 12 (19.7%) were homozygous for the 4/4 repeats. In the control group, the distribution was as follows: 42 (68.8%) five repeats homozygous, 12 (19.7%) heterozygous and 7 (11.5%) 4/4 homozygous. The number of carriers of the allele with four repeats was higher in the group of asthma patients, but the difference did not reach statistical significance with p ¼ 0.1712. Before treatment, no clinical differences were found among the three groups (Table 1). Conversely, the values obtained after the 6-month treatment period with montelukast were statistically significants, the worst data for all variables were found in the group 4/4 tandem repeats homozygous group. The response of heterozygous (4/5 repeats) patients was similar to the response of reference group (5/5 repeats), except for the b2-agonists use, which show intermediate values between both homozygous groups, although clearly better than those of the 4/4 patients (Table 1). Given that patients with genotypes 5/5 and 4/5 showed similar response to montelukast, we grouped together these patients and compared their response with the group of patients homozygous 4/4. There were neither demographic (age and sex) differences nor regarding to serum IgE levels or sensitisation to aero-allergens (pollen and dust mites) measured by means of prick test among both genotypic groups (Table 2). Moreover, both compared groups showed similar clinical features (FEV1, number of puffs of acting b2-agonist per day during the last month and number of exacerbations) before therapy (Figure 2 and attached table). After the LTs treatment period, patients with 4/4 repeats allele had 1.88 (SD ¼ 0.92) rate of asthma exacerbations in Table 1
859 the last month, with a mean reduction of 1.33 (SD ¼ 1.22), whereas for cases with at least one five repeat allele was 0.4 (SD ¼ 0.21) exacerbations with a reduction of 4.41 (SD ¼ 2.76), a very significant difference (p ¼ 0.001). The change in FEV1 was also more favourable in the group with at least a five repeats: 93% (SD ¼ 7.0) with an increase of 8.6% (SD ¼ 7.61) versus 80% and reduction of 1.4% (SD ¼ 8.04) (p ¼ 0.0006). Finally, the 4/4 repeats group needed more b2 rescue medication: 5.1 (SD ¼ 2.84) with a reduction of 0.55 (SD ¼ 1.66) versus 2.9 (SD ¼ 2.77) and a reduction of 4.3 (SD ¼ 3.42) in the 5/5 repeats group (p ¼ 0.0011).
Discussion The efficacy of asthma treatment depends on the control of environmental factors and also on pharmacological therapy. Nowadays, increasing evidence is supporting the concept of a tailored treatment for each patient. To reach this objective, an exact knowledge of genetic influences on drug response would be necessary.10,11 It is estimated that up to 70% of the variability of the response to asthma treatment is due to genetic conditions. Some authors previously reported the influence of the A-444C SNP of the LTC4 synthase gene to the response to CysLTR inhibitors,12,13 and a more recent study had discovered a better response to montelukast in patients bearing the less frequent genotypes of the MRP1 (rs119774) and ALOX5 (rs2115819).14 The allele with five tandem repeats (wild type) was the most frequently found in our study in agreement to other reports,6,9 the allelic frequency was 0.664 and the frequency of homozygous patients was 0.525. This polymorphism had been associated to some asthma phenotypes such as the severity,15 but never to asthma susceptibility. In addition to five and four repeats, three and six repeat alleles have been occasionally reported in some papers, but we did not find them in the present study. Prior to the LTRAs treatment period no significant difference was present with respect to FEV1, b2 agonist use, and exacerbation rate, between patients carrying or not the five repeats allele. Conversely, after the 6-month
Severity of asthma before and after 6-months treatment with montelukast.
ALOX5 genotype
(5/5) (n ¼ 32)
(5/4) (n ¼ 17)
(4/4) (n ¼ 12)
Sex distribution (M/F) Age (SD) IgE in KUI/L (SD) IgE rank
15/17 27.0 (13.8) 649.8 (473.3) 180–2300
8/9 27.9 (13.19) 556.8 (554.3) 290–2000
6/6 23.3 (8.7) 622.2 (511.7) 240–2000
Exacerbations in the last month mean (SD) Before 4.7 (2.7) After 0.4 (0.2)
5.0 (2.5) 0.4 (0.2)
3.22 (1.0) 1.88 (0.9)
FEV1 mean (SD) Before After
82% (8.1) 93% (7.3)
85% (7.0) 92% (6.9)
81% (13.5) 80% (9.8)
Puffs/24 h mean (SD) Before After
6.9 (3.0) 2.2 (1.8)
7.8 (3.0) 4.0 (3.9)
6.1 (1.6) 5.1 (2.8)
ARTICLE IN PRESS 860
J.J. Telleria et al.
Table 2 Age and sex distribution, IgE levels and sensitisation to aerorallergens of patients homozygous 4/4 and patients with at least one allele with five tandem repeats within the transcription factor-binding region of ALOX5 gene. Male/female
Age
IgE (IU/mL)
Prick test Pollen
Dust mites
Both
4/4 Mean SD
6/6
23.33 8.75
622.22 511.68
10 (83.3%)
3 (25%)
1 (8.3%)
5/4+5/5 Mean SD
23/26
27.21 13.23
625.34 479.13
44 (89.8%)
11 (22.4%)
6 (12.2%)
Before therapy Exacerbations
7
100%
P=n.s.
6
95%
5
90%
4
10 9 8 7 6 5 4 3 2 1 0
P=n.s.
85%
3 2
80%
1
75%
0
70% 5/5+5/4
B2 adrenergic Puffs / 24h
FEV1 (%)
4/4
5/5+5/4
4/4
P=n.s.
5/5+5/4
4/4
After therapy
7
B2 adrenergic Puffs / 24h
FEV1 (%)
Exacerbations 100%
P=0.001
6
95%
5
90%
4
P=0.0006
85%
3 2
80%
1
75%
0
70% 5/5+5/4
5/5+5/4
4/4
4/4
BEFORE THERAPY Exacerb. FEV1 (%) B2 / 24h 4/4
5/4+5/5
10 9 8 7 6 5 4 3 2 1 0
P=0.0011
5/5+5/4
4/4
AFTER THERAPY Exacerb. FEV1 (%) B2 / 24h
mean
3.22
81%
6.11
1.89
80%
5.56
SD
0.97
13%
1.62
0.93
10%
2.40
mean
4.76
83%
7.13
0.39
93%
2.71
SD
2.59
8%
3.00
0.59
7%
2.95
Figure 2
treatment with LTRAs, patients with at least one set of five repeats showed better clinical evolution outcomes than patients with 4/4 repeat alleles. In fact, among this last
group of patients, the average response was small or absent. The differences between the two groups were very high despite the small sample size.
ARTICLE IN PRESS ALOX5 genotype and response to montelukast In agreement with other studies,1,16 we found no differences between wild type homozygous (5/5 repeats) and heterozygous (4/5 repeats) suggesting that the wildtype allele would be dominant, at least for the studied phenotypes. Classically, three different mechanisms have been said to be involved in the influence of polymorphisms to the therapy response: (a) variations affecting drug metabolism, (b) variations related to mechanisms leading to adverse effects, and (c) variations in the therapeutic target or in the pathways activated by it.17 Our results are similar to other ones reported by Drazen et al.6 but they observed the correlation between the response to an ALOX5 inhibitor and the tandem repeat polymorphism in the promoter of the same gene. This fact is consistent with early studies supporting that addition and deletion variants in the ALOX5 gene promoter diminished the activity of promoter–reporter in vitro.5 Nevertheless, a later study supported that some mutant variants of the ALOX5 gene could also up-regulate the expression of this gene.14 ALOX-5 and montelukast are not directly related, since 5-LO initiates the LT synthesis pathway and montelukast is an antagonist of the receptors on which final products of the pathway, namely the CysLTs, exert their actions. Bearing in mind that ALOX-5 repeat polymorphism is a transcription factor binding site which modulates transcription of the reporter gene5 and thus the conversion of arachidonic acid into LTA4, it may be expected that patients with 4/4 repeat have a limited ability to initiate the cascade of LT synthesis and it could be hypothesized that in these patients LTs would probably not contribute much to the physiopathology of the asthma and thus their inhibition would be of very limited efficacy. Asthmatic patients could then be classified as LT-dependent, who would respond to LT pathway inhibitors, and LT-independent, who would not respond to those drugs. The importance of repeat polymorphism in the ALOX5 promoter on the response to LTRAs is confirmed. More polymorphisms related to the synthesis of LT could also be involved in the treatment response to LT inhibitors, but likely, the relative frequencies of those polymorphisms widely vary among different populations. From our data and given the frequency of allele 4 in our population, we should expect a ratio of bad responders to LT receptor inhibitors ranging between 12.3% and 18.9% (CI ¼ 95%). This ratio is not age or sex dependent, but could be higher in patients with moderate or severe asthma.15
Conflict of interest statement The authors declare that none of them have any financial or another sort of conflict of interest in relation to this study that may influence their interpretation of the results.
Acknowledgments This work has been partially sponsored by grants of Instituto de Salud Carlos III (PI05/1746) and Fundacion Sociedad Espan ˜ola de Alergia e Inmunologı´a Clı´nica (2005).
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